Template-directed synthesis of oligoguanylates in the presence of metal ions

doi:10.1016/0022-2836(80)90338-1

Journal of Molecular Biology

Volume 144, Issue 4, 25 December 1980, Pages 579-585

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Abstract

We have studied the condensation reaction of ImpG^† on a poly(C) template, in the presence of various metal ions. With Mg²⁺ as co-catalyst we confirmed that Pb²⁺ and Zn²⁺ are effective catalysts. A catalytic effect was also observed for Bi³⁺, Sb³⁺ and Mn²⁺. Bi³⁺ and Sn²⁺, like Pb²⁺, favored the formation of 2′-5′ linkages. With Mn²⁺ a rather complex mixture of oligomers is formed, some of which contain pyrophosphate linkages. None of the metal ions investigated behaved like Zn²⁺ in favoring the formation of the naturally occurring 3′-5′ linkages.

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Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C, U) templates
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Poly(C, U) random copolymer templates direct the oligomerization of 2-MeImpG^† and 2-MeImpA, resulting in the production of a variety of oligo(G, A)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpA, and decreases for both monomers as the uracil content of the template increases. The relatively poor incorporation of adenine is partly due to an intrinsically less efficient incorporation reaction, and partly due to the masking of uracil sites by G · U non-complementary pairing. The efficiency of adenine incorporation can be improved by decreasing the concentration of 2-MeImpG and increasing the concentration of 2-MeImpA in the reaction mixture.
The oligomeric product distribution can be characterized in detail using highpressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phospho-diester-linkage isomerism. The 3′ → 5′ regiospecificity of monomer addition to template-bound oligomers is lower for 2-MeImpA than for 2-MeImpG. The presence of an adenine residue at the 2′(3′) terminus of the acceptor strand lowers the regiospecificity of 2-MeImpA addition even further.
Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C). An RNA polymerase model
1982, Journal of Molecular Biology
(Guanosine 5′-phosphor)-2-methylimidazolide (2-MeImpG), unlike guanosine 5′-phosphorimidazolide (ImpG), undergoes an efficient, buffer-independent, template-directed oligomerization in the presence of poly(C) at pH values above 7.6. The reaction occurs in a Watson-Crick double helix and yields predominantly 3′-5′-linked oligomers up to the 50-mer in above 80% yield. Synthesis proceeds in the 5′ → 3′ direction and has high fidelity in the sense that nucleotides other than G are not incorporated significantly into oligomers. Under some conditions, oligomers corresponding to approximately one and two turns of the helix are obtained in higher yield than somewhat longer or somewhat shorter oligomers.
In the protonated triple-helical structure formed below pH 7, the efficiency of the oligomerization is much lower. Oligomers up to about the 10-mer are obtained. The most abundant products are “capped” at the 5′ terminus with a GppG pyrophosphate group.
Catalysis of accurate poly(C)-directed synthesis of 3′-5′-linked oligoguanylates by Zn<sup>2+</sup>
1980, Journal of Molecular Biology
Zn²⁺ is an efficient catalyst for the oligomerization of guanosine 5′-phosphorimidazolide on a polycytidylic acid template. Up to 75% of the input ImpG^† is converted to oligomers with a mean chain length up to 10. Material longer than (pG)₃₀ can be detected. The oligomeric products are predominantly 3′-5′-linked.
If poly(C) is incubated with ImpG and an equimolar quantity of the 5′-phosphorimidazolides of adenosine, uridine or cytidine, in the presence of Zn²⁺, ImpG is incorporated at least 200 times more efficiently than “incorrect” nucleotides.

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^☆: Support for this study came from National Aeronautics and Space Administration grant no. NGR 05-067.

^†: Abbreviations used: G, guanosine; Gp, guanosine 2′(3′)-phosphate; pG, guanosine 5′-phosphate; Gp!, guanosine cyclic 2′,3′-phosphate; ImpG, guanosine 5′-phosphorimidazolide; ImpA, adenosine 5′-phosphorimidazolide; pGp, 5′-phosphoguanosine 2′(3′)-phosphate; G²pG, guanylyl-[2′-5′]-guanosine; G³pG, guanylyl-[3′-5′]-guanosine; ImpGpG, 5′-phosphorimidazolide of GpG; (pG)_n (n = 2,3, …), oligomers of pG; GppG, P₁,P₂-diguanosine 5′-diphosphate; GppGpG, P₁,P₂-guanosine 5′-diphosphate derivative of guanylyl guanosine; NH₂pG, guanosine 5′-phosphoramidate; (pG)₄₊, tetramer and higher oligoguanylates with 5′-terminal phosphate; poly(C), polycytidylic acid; poly(U), polyuridylic acid.

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Journal of Molecular Biology

Template-directed synthesis of oligoguanylates in the presence of metal ions☆

Abstract

J. Mol. Biol

Tetrahedron

errata

Science

J. Mol. Biol